The relative deacylation of microsomal phospholipid molecular species was reexamined. Microsomal membranes were prepared from the livers of rats injected, over a period of 20 h, with perdeuterated ethanol. The phosphatidylcholine and phosphatidylethanolamine were isolated by thin-layer chromatography of the total lipid extracts and the distribution of deuterium among the molecular species of the diacylglycerol moieties was determined by reversed-phase high pressure liquid chromatography in combination with chemical ionization mass spectrometry. Deuterium was found to be incorporated into newly formed glycerol and newly synthesized palmitic and stearic acids (4-22% replacement) which were distributed throughout the molecular species in proportion to their relative rates of turnover (10-45% replacement). Within each unsaturation class, the palmitoyl species were labelled more extensively than the corresponding stearoyl species. Following in vitro incubation of the membranes with 10 mM Ca2+ at pH 8.5, there occurred a rapid degradation of the phosphatidylethanolamines (to 60% of control values after 90 min), while the phosphatidylcholines remained essentially unaffected. The various molecular species of the phosphatidylethanolamines were degraded linearly and in proportion to their masses. The deuterium content of the phosphatidylethanolamine and phosphatidylcholine remained constant throughout the incubation. It was concluded that under the present experimental conditions all molecular species of phosphatidylethanolamine, both old and newly synthesized, are equally accessible to the endogenous phospholipase.